Primary Aldosteronism: New Answers, New Questions.

There have been 2, and possibly 3, major questions for primary aldosteronism (PA) answered at least in principle over the past 5 years. The first is that of somatic mutations underlying the majority of aldosterone producing adenomas. The second is the extension of our knowledge of the genetics of familial hypertension, and the third the role of renal intercalated cells in sodium homeostasis. New questions for the next 5 years include a single accepted confirmatory/exclusion test; standardisation of assays and cut-offs; alternatives to universal adrenal venous sampling; reclassification of 'low renin hypertension'; recognition of the extent of 'occult' PA; inclusion of low-dose mineralocorticoid receptor antagonist in first-line therapy for hypertension; and finally, possible resolution of the aldosterone/inappropriate sodium status enigma at the heart of the cardiovascular damage in PA.

Primary aldosteronism: are we missing the wood for the trees?

The prevalence of primary aldosteronism (PA) is around 10% of hypertensives, with markedly increased risk of cardiovascular damage compared with age-, sex- and BP-matched essential hypertension (EH). Currently, if hypertension is present in 20% of the population, PA will account for 2%; of these PA patients only 1% are ever screened, let alone diagnosed and treated, and the remaining 99% suboptimally treated, if at all. Mineralocorticoid receptor (MR) antagonists are effective in lowering BP, uniquely vasoprotective and safe when titrated to effect in EH. In resistant hypertension (BP elevated despite 3 or more conventional agents, including a diuretic), which constitutes 20-30% of EH, addition of a low dose MR antagonist reproducibly produces BP lowering of 20-30 mm Hg. Two thirds of PA is unilateral, and normally treated by MR antagonists; in unilateral PA surgery is recommended, but there are also studies reporting MR antagonist therapy to be noninferior over the longer term. There thus seems to be a very strong case for including a low dose MR antagonist in first-line therapy for new hypertension, given its utility and safety across EH, its particular efficacy in resistant hypertension, and its specific benefits for the 99% of subjects with occult PA. We do not have the resources to diagnose PA, but we do have the wherewithal to treat it.

Distinguishing the antihypertensive and electrolyte effects of eplerenone.

In two clinical trials on the antihypertensive effects of the mineralocorticoid receptor antagonist eplerenone 397 essential hypertensives were dose titrated (50, 100, and 200 mg/d) over successive 4-wk periods until they reached target blood pressure levels. Of the total, 44% reached target on 50 mg/d, 17% on 100 mg/d, and 19% on 200 mg/d, with 20% failing to do so despite stepwise dose increases. At each dose level, those who reached target (responders) were compared with those who did not (nonresponders), with three major findings. First, at each dose level, the blood pressure fall in responders (systolic, 16-20 mm Hg; diastolic, approximately 15 mm Hg) was markedly more than mean values in nonresponders (systolic, 2-5 mm Hg; diastolic, 1-3 mm Hg). Second, sensitivity to eplerenone varied widely across the population studied in terms of blood pressure reduction. Third, there was no difference in plasma [K+] levels between responders and nonresponders at any dose level. We interpret these data as evidence for the major antihypertensive effect of eplerenone being via mechanisms other than those involving epithelial electrolyte and fluid transport. The modest (< or =0.2 mEq/liter at 200 mg/d) mean elevation in plasma [K+] suggests that titration to effect rather than forced titration may minimize the risk of hyperkalemia, even where relatively high (100-200 mg/d) doses of the specific mineralocorticoid receptor antagonist eplerenone may ultimately be required.

Cardiac steroidogenesis in the normal and failing heart.

The present study explores the possibility of local de novo aldosterone production in normal and failing hearts (human and mouse) and the regulation of such putative cardiac steroidogenesis. Total RNA was isolated from human tissue from failing hearts taken at the time of cardiac transplantation, from normal hearts obtained at autopsy, and from normal and pressure-overloaded mouse hearts. Vascular smooth muscle cells from human artery and vein were also analyzed. RNA was reverse transcribed and probed with specific primers for side-chain cleavage enzyme (CYP11A), 3beta-hydroxysteroid dehydrogenase, aldosterone synthase (CYP11B2), 11beta-hydroxylase (CYP11B1), steroidogenic factor-1, and steroid acute regulatory protein. CYP11A, 3beta-hydroxysteroid dehydrogenase-2, and steroid acute regulatory protein were expressed at modest levels in all tissues examined in both mouse and human. In failing human heart, CYP11B1 and CYP11B2 were detected in some samples, in contrast with normal hearts, which expressed neither; in the mouse heart steroidogenic factor-1 was detected, but neither CYP11B1 nor CYP11B2 was found. Steroidogenic factor-1 was detected in no human heart sample tested after 40 cycles of PCR. Although the expression of some steroidogenic genes can be detected in the heart, the likelihood of physiologically relevant levels of aldosterone production by the normal heart is very low. The exact cellular location of steroid synthesis in the failing human heart remains to be established.

Experimental cardiac fibrosis: differential time course of responses to mineralocorticoid-salt administration.

The rapid (1-4 h) responses of epithelial target tissues to mineralocorticoids contrast with the days/weeks apparently required for responses in the cardiovascular system. The present study explores the time course and pattern of early events leading to cardiac fibrosis in the mineralocorticoid-salt rat model. Uninephrectomized rats were given deoxycorticosterone (20 mg, sc, weekly) plus 0.9% NaCl/0.3% KCl to drink and were killed at 2, 4, 8, 16, and 32 d. Type III collagen increased progressively from d 2, and blood pressure from d 4, with 4 and 8 d rats showing marked perivascular inflammatory cell infiltration. Apoptosis was also noted in perivascular areas at 4 and 8 d and in scar areas at 8, 16, and 32 d. Elevation of mineralocorticoid hormone levels inappropriate for salt status thus provokes a series of changes in cardiac vessels and myocytes leading to increased collagen deposition. When mineralocorticoid levels are elevated acutely by bolus injection, changes are discernible after 2 d, in contrast with previous infusion studies in which 3-4 wk were required for measurable changes.

The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action.

Aldosterone plays a major role in regulating sodium and potassium flux in epithelial tissues such as kidney and colon. Recent evidence suggests that serum- and glucocorticoid-regulated kinase (SGK) is induced by aldosterone and acts as a key mediator of aldosterone action in epithelial tissues. Induction of SGK messenger RNA (mRNA) has previously been shown within 30 min of addition of supraphysiological doses of aldosterone to Xenopus A6 cells and within 4 h in rat kidney in vivo. In this study we determined the time course of SGK induction, at doses of aldosterone in the physiological range, in rat kidney and colon, using Northern and Western blot analyses and in situ hybridization and determined concurrent changes in urinary sodium and potassium excretion by Kagawa bioassay. On Northern blot analysis, SGK mRNA levels were significantly elevated in both kidney and colon 60 min after the injection of aldosterone. SGK protein in late distal colon was significantly elevated 2 and 4 h after aldosterone treatment. In situ hybridization showed SGK mRNA to be induced in renal collecting ducts and distal tubular elements in both cortex and medulla by doses of aldosterone of 0.1 microg/100 g BW or more within 30 min of steroid treatment. Significant changes in urinary composition were similarly seen with an aldosterone dose of 0.1 microg/100 g BW from 90 min after aldosterone injection. The early onset of SGK induction in kidney and colon and the correlation with urinary changes in terms of both time course and dose response suggest that SGK plays an important role in mediating the effects of aldosterone on sodium homeostasis in vivo.

GRKO mice express an aberrant dexamethasone-binding glucocorticoid receptor, but are profoundly glucocorticoid resistant.

The introduction of a targeted insertion mutation into exon 2 of the gene coding for the glucocorticoid receptor (GR) enabled production of glucocorticoid receptor knock-out (GRKO) mice. GRKO mice on a C57BL/6/129sv mixed genetic background show a variable phenotype, with 90% of -/- mice dying at birth with respiratory insufficiency but 10% of mutant mice surviving to maturity. To investigate the possibility of residual GR expression in surviving GRKO mice we have measured binding of the synthetic glucocorticoid dexamethasone in tissue extracts from adrenalectomized mice. High affinity binding of dexamethasone in protein extracts of liver, kidney, lung and brain from adult GRKO mice is found at levels 30-60% those in wild-type mice, with heterozygotes (+/-) having intermediate levels. PCR and ribonuclease protection analysis showed comparable levels of GR mRNA on the 3' side of the gene-targeted insertional mutation in exon 2 of the GR gene, with almost no GR mRNA detected from exons 1 and 2 on the 5' side of the gene-targeted insertional mutation. Western blot analysis using a C-terminal specific GR antibody detects a 39 kDa GR fragment in extracts from adult GRKO mice. Despite the evidence for expression of a ligand-binding domain fragment of the glucocorticoid receptor these mice are profoundly glucocorticoid resistant, with elevated levels of plasma ACTH and corticosterone. Thymocytes from adult and fetal GRKO mice are resistant to dexamethasone-induced apoptosis and cultured fetal hepatocytes from GRKO mice are completely refractory to glucocorticoid induction of the gluconeogenic enzyme glucose-6-phosphatase. Thus although the surviving adult homozygous GRKO mice express a dexamethasone-binding GR fragment, their classic target tissues remain profoundly glucocorticoid insensitive.

Non-genomic actions of aldosterone: role in hypertension.

There is universal acceptance of the existence of rapid, non-genomic effects of aldosterone, although their physiological relevance and potential importance in hypertension are not yet clear. What has emerged over the year under review is that at least some of such rapid non-genomic effects of aldosterone may be mediated by the activation of the classical intracellular mineralocorticoid receptor, rather than a putative membrane receptor. The post-receptor mechanisms of rapid aldosterone action appear variously to involve protein kinase C, calcium, cyclic adenosine 3', 5'-monophosphate and inositol 1, 4, 5-triphosphate, with downstream effects on a variety of ion pumps and channels.

Dynamin II regulates hormone secretion in neuroendocrine cells.

The dynamin family of GTP-binding proteins has been implicated as playing an important role in endocytosis. In Drosophila shibire, mutations of the single dynamin gene cause blockade of endocytosis and neurotransmitter release, manifest as temperature-sensitive neuromuscular paralysis. Mammals express three dynamin genes: the neural specific dynamin I, ubiquitous dynamin II, and predominantly testicular dynamin III. Mutations of dynamin I result in a blockade of synaptic vesicle recycling and receptor-mediated endocytosis. Here, we show that dynamin II plays a key role in controlling constitutive and regulated hormone secretion from mouse pituitary corticotrope (AtT20) cells. Dynamin II is preferentially localized to the Golgi apparatus where it interacts with G-protein betagamma subunit and regulates secretory vesicle release. The presence of dynamin II at the Golgi apparatus and its interaction with the betagamma subunit are mediated by the pleckstrin homology domain of the GTPase. Overexpression of the pleckstrin homology domain, or a dynamin II mutant lacking the C-terminal SH3-binding domain, induces translocation of endogenous dynamin II from the Golgi apparatus to the plasma membrane and transformation of dynamin II from activity in the secretory pathway to receptor-mediated endocytosis. Thus, dynamin II regulates secretory vesicle formation from the Golgi apparatus and hormone release from mammalian neuroendocrine cells.

Aldosterone and the heart.

Classically, aldosterone is a steroid hormone secreted from the adrenal cortex, which acts on kidney, colon and sweat/salivary glands to promote unidirectional sodium transport. Currently, there is excellent experimental evidence for aldosterone acting directly on the central nervous system to raise blood pressure, and on the heart to cause cardiac hypertrophy and fibrosis. In addition, there is emerging evidence for aldosterone synthesis in the heart, and for as yet unexplained benefits of aldosterone antagonism in the treatment of cardiac failure.

Aldosterone and mineralocorticoid receptors: orphan questions.

Classically, mineralocorticoid receptors (MR) are activated by aldosterone to promote unidirectional transepithelial sodium transport. Activation of MR in nonepithelial tissues has been shown to elevate blood pressure (central nervous system; CNS) and to cause hypertrophy and fibrosis (heart). For both epithelial and nonepithelial tissues, there remain a variety of questions regarding MR which are not only unanswered but also essentially not addressed. Seven such questions include: (1) how the physiologic glucocorticoids (cortisol and corticosterone) can mimic aldosterone action in epithelial MR, but act as antagonists in the heart and AV3V region; (2) how salt facilitates the nonepithelial, pathophysiologic effects of aldosterone; (3) how aldosterone activates unprotected AV3V MR in the face of orders of magnitude higher circulating glucocorticoid concentrations; (4) how unprotected nonepithelial MR act as "always occupied" receptors in guinea pigs and other species; (5) how, when 11beta hydroxysteroid dehydrogenase type 2 is active, epithelial MR occupied by physiologic glucocorticoids appear transcriptionally inactive; (6) how aldosterone activates epithelial MR in the face of approximately 103-fold higher glucocorticoid levels, plasma binding and 11beta hydroxysteroid dehydrogenase type 2 activity notwithstanding; and (7) how aldosterone produces changes in urinary [K+] before [Na+].

Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro.

The telomere DNA polymerase (telomerase) and the tumor suppressor protein p53 are frequently associated with human cancers, and activation of telomerase and inactivation of p53 involved in cancer cell immortalization. In this report, we demonstrate a direct interaction of telomerase with p53 in the nuclear lysates of human breast cancer cells, and with recombinant human p53, by affinity chromatography and immunoprecipitation. On activity criteria, the interaction is between the carboxyl-terminal region of p53 and a region close to the amino-terminus of human telomerase-associated protein 1 (hTEP1). Incubation of recombinant p53 with nuclear telomerase extracts results in inhibition of telomerase activity, with the C-terminal region of p53 being essential for inhibition. This effect is not mediated by binding to telomerase substrate DNA, but requires the region near the N-terminus of hTEP1, in that a synthetic peptide derived from this region of hTEP1 similarly inhibits telomerase activity. Together, these in vitro interactions between telomerase and p53 suggest that the activity of telomerase may be regulated by p53, down-regulation of which in turn would favor up-regulation of telomerase activity in cancer cell development.

Growth factors and extracellular signal-regulated kinase in vascular smooth muscle cells of normotensive and spontaneously hypertensive rats.

OBJECTIVE: Transforming growth factor-beta1 (TGF-beta1) stimulates vascular smooth muscle cell growth in spontaneously hypertensive rats (SHR), but inhibits cell growth in normotensive Wistar- Kyoto (WKY) rats. The present study was undertaken to test the hypothesis that TGF-beta1 might differentially modulate the activities of mitogen-activated protein (MAP) kinase family members (ERK, JNK and p38) in vascular smooth muscle cells of SHR and WKY rats. METHODS: MAP kinase activity was measured from cultured vascular smooth muscle cells in response to TGF-1 by specific substrate phosphorylation of myelin basic protein, GST-c-Jun and GST-ATF2. RESULTS: Exposure of cultured vascular smooth muscle cells from SHR or WKY rats to TGF-beta1 resulted in a marked increase in the activity of ERK, but not of JNK or p38. The increase of ERK activity stimulated by TGF-beta1 appeared similar in time course and extent in both WKY and SHR cells, with increased activity peaking at 15 min of incubation. Epidermal growth factor (EGF) also stimulated the activity of ERK, in both WKY and SHR cells, but nor of JNK or p38, with stimulation of ERK activity by EGF occurring more rapidly in SHR cells than in those from WKY rats. Co-incubation of SHR cells with TGF-beta1 and EGF showed additive effect on ERK activity. CONCLUSIONS: The results provide the first evidence that TGF-beta1 activates ERK in vascular smooth muscle cells of both normotensive and hypertensive rats. The matching response of ERK activation to TGF-1 in SHR cells suggests that the MAP kinase-signaling pathway remains largely unchanged in the regulation of vascular smooth muscle growth by TGF-1 in spontaneously hypertensive rats.